New York State Department of Health - Wadsworth Center Laboratory of Environmental Biology NYS ELAP Laboratory ID 10765

Transcription

1 New York State Department of Health - Wadsworth Center Laboratory of Environmental Biology NYS ELAP Laboratory ID Division of Environmental Health Sciences Albany, New York NYS DOH LEB-606 Identification of Bile Tolerant Gram Negative Bacteria in Medical Marijuana Products Page 1 of 14

2 Table of Contents 1.0. Scope and Application Summary of the Method Definitions Health and Safety Warnings Shipping Conditions, Receiving, Preservation and Storage Interferences Apparatus and Materials Quality Control/Assurance (Laboratories must conform to sections of Standard Methods for the Examination of Water and Wastewater.) Procedure Data Acquisition, Reduction, Analysis, Calculations, Acceptance Criteria and Documentation Method Performance Waste Management/Pollution Prevention References Appendices Page 2 of 14

3 1.0. Scope and Application 1.1. This method, NYS DOH LEB-606, describes methods for detecting and identifying Bile Tolerant Gram Negative bacteria (ELAP Method ID 9987) in medical marijuana samples as required in Title 10 (Health), Subpart and Chapter XIII, Section of the official Compilation of Codes, Rules, and Regulations, of the State of New York. It is used as a follow-up to NYS DOH LEB-604 section 9.2, and applies to sample enrichments showing growth in Enterobacteria Enrichment Broth Mossel Protocols for the identification of these organisms in samples of medical marijuana products can be found in the NYS DOH LEB-600 series. See Appendix A for Medical Marijuana Microbial Testing Plan flowcharts Summary of the Method 2.1. Medical marijuana samples showing growth in Enterobacteria Enrichment Broth Mossel are subcultured onto Violet Red Bile Glucose Agar and incubated at C for hours. Bacterial colonies are transferred to Trypticase Soy Agar and identified using a commercially available identification system, e.g. API 20E identification strips. Samples producing bacterial colonies on Violet Red Bile Glucose Agar that are identified as Enterobacteriaceae, aeromonads or pseudomonads are reported as positive for Bile Tolerant Gram Negative organisms Definitions 3.1. Bile Tolerant Gram Negative organisms are defined as bacteria that grow on VRBGA plates under growth conditions prescribed herein. Examples include members of the Enterobacteriaceae, aeromonads and pseudomonads TSA stands for Trypticase Soy Agar 3.3. EEBM stands for Enterobacteria Enrichment Broth Mossel 3.4. VRBGA stands for Violet Red Bile Glucose Agar 4.0. Health and Safety Warnings 4.1. Microbiological analyses involve the culturing of potentially pathogenic organisms All microbiologically contaminated materials, including media, shall be autoclaved after use Laboratory equipment and benches shall be disinfected before and after use with a minimum concentration of 70% ethanol Mouth pipetting is prohibited All accidents, particularly those which may result in infection, shall be reported according to laboratory specific policies and procedures Laboratory safety procedures shall be followed at all times. Regulations required by federal, state and local government agencies shall be implemented and followed The analyst must be familiar with any possible hazards from the reagents and standards used for sample preparation and analysis Always follow guidelines listed in safety data sheets (SDS) for proper storage, handling, and disposal of samples, solvents, reagents, and standards. SDS are Page 3 of 14

4 located within the laboratory. These guidelines must be made available to all personnel involved in microbiological analyses Appropriate lab coat, safety glasses and gloves must be worn when performing standard or sample preparations, working on instrumentation, disposing of waste, and cleaning laboratory equipment Shipping Conditions, Receiving, Preservation and Storage 5.1. Sample shipping conditions - The medical marijuana products from the Registered Organizations (ROs) are shipped as per manufacturer s specifications and must adhere to all regulatory requirements Sample Receipt - Medical marijuana products from the RO are received, verified and documented ensuring that method, regulatory and Accreditation Body requirements are met. All medical marijuana products must be stored under the conditions based on the manufacturer s recommendation. The storage is documented Method holding times Not determined Preservation Samples diluted in PBST that are not required for analyses are stored refrigerated until it has been determined that they are not needed for additional microbiological evaluation Presumptive positive sample enrichments are stored refrigerated until microbiological evaluation/identification is completed Storage Samples are analyzed upon receipt If storage is required, samples are maintained at room temperature in a secure location Interferences 6.1. The presence of spreading colonies or confluent growth can interfere with accurate colony identification Apparatus and Materials 7.1. Equipment Incubator, C Automatic pipetters and sterile tips Sharpie or equivalent Sterile inoculating loops, 10µL 7.2. Reagent and Chemicals VRBA plates TSA plates supplied Oxidase test, e.g., DrySlide Oxidase Slides, BD BBL cat. no Commercially available bacterial identification system (e.g., API 20E test strips, biomérieux cat. No ) Page 4 of 14

5 Any reagents required for the bacterial identification system 7.3. Forms Bile Tolerant Gram Negative Identification Result Sheet (e.g. LEB-RS- 606A, Appendix B) Quality Control/Assurance (Laboratories must conform to sections of Standard Methods for the Examination of Water and Wastewater.) 8.1. Method Detection Limits Not determined Calibration and Standardization Incubator temperatures shall be observed and recorded twice daily, separated by at least 4 hours Temperature of the C walk-in is recorded If the incubator temperature does not stay within C, laboratory specific corrective actions are followed. Analytical results are invalidated if the incubator temperature exceeds 35.0 C Temperatures of the cold room and refrigerators are observed and recorded at least once daily If the cold room or refrigerator does not stay within 1-8 C, laboratory specific corrective actions are followed The optimum temperature range for a refrigerator is 1-4 C If the cold room or refrigerator was in a defrost cycle at the time that the temperature was recorded, and the temperature does not reach 8 C, re-testing of media is not required Media may be re-tested for quality, depending on the number of degrees and the amount of time that the cold room temperature was out of compliance, at the discretion of the laboratory Max/min temperatures are recorded when daily temperature measurements are not possible, such as on holidays and weekends Thermometers must be calibrated as prescribed by the Accreditation Body and in accordance with relevant regulations and standards Sterility of disposable loops and spreaders is determined as prescribed by the Accreditation Body and in accordance with relevant regulations and standards Micropipetters are calibrated as prescribed by the Accreditation Body and in accordance with relevant regulations and standards Quality Control Invalidate lot of media if tests are not in accordance with acceptance criteria as prescribed by the Accreditation Body and in accordance with relevant regulations and standards Acceptability of supplies is tested as prescribed by the Accreditation Body and in accordance with relevant regulations and standards The use test for deionized water is performed annually, when cartridges are changed, or repairs are made to the deionized water systems and as Page 5 of 14

6 prescribed by the laboratory, Accreditation Body and in accordance with relevant regulations and standards Liquid media shall be stored in tightly-capped bottles in the dark at 4 C for up to 3 months from the date of preparation Agar plates can be used for 2 weeks if stored refrigerated in plastic bags and in the dark Agar plates can be used after 2 weeks storage if ongoing QC demonstrates no loss in selectivity or growth promotion Corrective/Preventive Actions The laboratory will initiate non-conformances and/or corrective/preventive actions in accordance with laboratory specific procedures and as prescribed by the Accreditation Body and in accordance with relevant regulations and standards Procedure 9.1. Subculture Aseptic technique is used for all procedures Aseptic technique can be found in a general microbiology textbook or on-line For each turbid EEBM sample enrichment produced according to NYS DOH LEB-604, section 9.2, remove three VRBGA plates from the cold room and warm to room temperature while drying in the biological safety cabinet Two plates will be used for the sample enrichment and one for the corresponding matrix spike Use an inoculating loop to streak sample from the turbid EEBM sample enrichment onto two VRBGA plates for colony isolation Use an inoculating loop to streak the corresponding turbid EEBM matrix spike enrichment onto one VRBGA plate for colony isolation Once the samples have dried, invert the plates and incubate at C for hours Do not stack the plates more than four high After incubation, record colony characteristics and growth for the sample plates as growth-positive (bacterial colonies are present) or negative (bacterial colonies are absent) (e.g. LEB-RS-606A) If the sample is positive on VRBGA, proceed to If there is no growth on the VRBGA plates, the sample is negative for the presence of BTGN bacteria After incubation, record colony characteristics and growth of the matrix spike plates as growth-positive (presence of bacterial colonies) or negative (absence of bacterial colonies) and colony characteristics (e.g. LEB-RS- 606A) If the sample result is negative on VRBGA, it is not necessary to identify colonies on a positive matrix spike sample plate that show morphology typical of K. pneumoniae ATCC Page 6 of 14

7 Typical K. pneumoniae ATCC appears as shiny purple/pink colonies If the matrix spike is positive on VRBGA, proceed to If the matrix spike result is negative on VRBGA, the test results are invalidated If the positive and negative controls for aerobic plate counts and mold plate counts meet QC criteria (see NYS DOH LEB-605 and NYS DOH LEB-609), results are considered valid in the absence of turbidity in the matrix spikes If the positive and negative controls for aerobic plate counts and mold plate counts do not meet QC criteria (see NYS DOH LEB-605 and NYS DOH LEB-609), results are considered invalid and the analyses must be repeated Additional testing using USP methods designed to decrease inhibitory effects of product that could result in growth in matrix spike samples may be undertaken using archived samples, at the discretion of the Laboratory Colony Identification Streak well-isolated colonies having distinct morphologies from positive sample VRBGA plates onto TSA plates that have been warmed to room temperature and dried in a biological safety cabinet Bacterial colonies can be selected from either of the two VRBGA sample plates If growth is confluent on the VRBGA plates, re-streak for isolation of individual colonies and proceed with At a minimum, streak well-isolated colonies showing characteristics typical of K. pneumoniae ATCC from the positive matrix spike sample onto TSA plates that have been warmed to room temperature and dried in a biological safety cabinet If growth is confluent, re-streak for isolation of individual colonies and proceed with Invert TSA plates and incubate at C for hours Do not stack more than four high After incubation, choose a well-isolated colony from each TSA plate and proceed with a commercially available identification system, e.g. the API Identification Test Strip method, to identify the organism using API 20E Test Strips Attach all API 20E Identification Results sheets (or relevant paperwork from other identification systems) to the Bile Tolerant Gram Negative Bacteria Identification Results Sheet (e.g. LEB-RS-606A). Page 7 of 14

8 10.0. Data Acquisition, Reduction, Analysis, Calculations, Acceptance Criteria and Documentation Record the accession number, analyst initials, VRBGA lot date, start and end dates and times, TSA lot date, start and end dates and times, bacterial identification system lot and expiration dates, start and end dates and times, colony morphology, source of colony (matrix spike or sample), colony identification and results (e.g. LEB-RS-606A) Report samples showing bacterial growth on VRBGA that result in identification of bile tolerant gram negative species as positive for Bile Tolerant Gram Negative organisms Report samples showing growth on VRBGA that do not result in identification of bile tolerant gram negative species as negative for Bile Tolerant Gram Negative organisms Report as negative samples showing no growth in EEBM or VRBGA Invalidate the test results for samples lacking growth in the matrix spike at any point in the analysis or from which bile tolerant gram negative bacteria were not identified See section Method Performance Demonstration of Capability Prior to acceptance and use of this method for data reporting, a satisfactory initial demonstration of capability (DOC) is required. Thereafter, an ongoing DOC is to be performed annually An initial DOC shall be made prior to using any method, and at any time there is a change in instrument type, personnel or method or any time that a method has not been performed by the laboratory or analyst in a twelve (12) month period All DOCs shall be documented, and all data applicable to the demonstration shall be retained and readily available at the laboratory. Consult state regulations and standards for additional information on performing a DOC for microbiological contaminants Consult relevant standards, regulations and Accreditation Body requirements for additional information on performing DOCs for microbial contaminants Laboratory Detection Limits Detection limits are determined in accordance with relevant standards, regulations and accreditation body requirements. Page 8 of 14

9 12.0. Waste Management/Pollution Prevention It is the responsibility of the laboratory to comply with all federal, state and local regulations governing waste management, particularly the hazardous waste identification rules and land disposal restrictions Bacterial/fungal cultures and contaminated or potentially contaminated disposable materials are disposed of in biohazardous waste cans and autoclaved prior to discarding Dispose of non-hazardous water waste in the laboratory sink followed by flushing with tap water Dispose of glassware in appropriately labeled boxes. Be sure that, whenever possible, the glass has been thoroughly rinsed and is contaminant-free before disposal Consult federal, state and local regulations for additional information or for information on the disposal of products not described in this method References United States Pharmacopeia. USP38-NF33, The United States Pharmacopeial Convention, General chapters <61>, <62>, <1111> API 20E Test Strips Instructions for Use, biomérieux NYS DOH LEB-604, Microbial Presence/Absence Tests for Medical Marijuana Samples Page 9 of 14

10 14.0. Appendices Appendix A Flowcharts Page 10 of 14

11 Page 11 of 14

12 Page 12 of 14

13 Page 13 of 14

14 Appendix B - Forms Bile Tolerant Gram Negative Bacteria Identification Results Sheet (LEB-RS-606A) Incubate VRBGA for hours and TSA for hours (30-35 C Incubator, E552) Accession Number: Analyst Initials: VRBGA Start Date/Time: VRBGA End Date/Time: VRBGA Lot Date: Sample Result: M.S. Result: TSA Start Date/Time: TSA End Date/Time: TSA Lot Date: API 20E Start Date/Time: API 20E End Date/Time: Source (M.S. or Sample), Colony Morphology and API 20E Colony Identification All API 20E Result Sheets are attached. EEBM = Ent. Enrichment Broth Mossel, VRBGA = Violet Red Bile Glucose Agar, TSA = Trypticase Soy Agar K. pneumoniae ATCC 13883, used as a matrix spike, is typically pink/purple and shiny on VRBGA agar. Reviewed by Date Page 14 of 14